Kinetics, anion binding and mechanism of Co(II)‐substituted bovine muscle carbonic anhydrase

Abstract

The binding of N; to Co(I1)-substituted bovine carbonic anhydrase 111 was measured at various pH values by spectrophotometric titrations. The apparent Ki values were found to increase with pH in the studied range between pH 5.8 and 8.9. The inhibition of C 0 2 hydration by N; was found to be essentially uncompetitive at all investigated pH values (pH 6.3 -8.9). The Ki values for the inkbition of k,,, are much smaller than those obtained in the spectrophotometric titrations indicating that an enzyme form with a high affinity for N;, presumably having a metal-bound H20, accumulates in the steady state at saturating COz concentrations. Assuming that the low pH limit of Ki = 9 pM for the inhibition of k,,, represents the affinity of N; for the Co(I1)-OHz form, a pKa value near 5 can be estimated for Co(I1)-bound water from the pH dependence of N; binding in the absence of c02.Measurements of time-resolved absorption spectra during C 0 2 hydration in the presence of a low N; concentration showed the transient appearance of the characteristic spectrum of the enzyme-N; adduct clearly demonstrating the accumulation in the steady state of an enzyme form with a high affinity for N;. In similar experiments without inhibitor the transient formation of a spectral form corresponding to a Co(I1)-OH2 species has been demonstrated. This spectral form is rather featureless lacking the absorption maxima at 618 nm and 640 nm characteristic of the Co(I1)-OH-species. Our results strongly support the hypothesis that the rate-limiting step in C 0 2 hydration catalyzed by carbonic anhydrase I11 is the protolysis of metal-bound water.Three cytosolic isoenzyme forms of carbonic anhydrase are known to occur in higher vertebrates [l]. They are genetically distinct and have different kinetic and inhibitorbinding properties but are structurally homologous. The highactivity carbonic anhydrase 11, with a maximal C 0 2 hydration turnover number of lo6 s -l at 25"C, has been studied extensively. Two functional groups withpK, values near 7 at normal ionic strengths are thought to be involved in catalysis. These groups are presumably a metal-bound water molecule and the imidazole group from His-64 [I]. Kinetic studies have shown that the interconversion between C 0 2 and HCO; is temporally separate from the transfer of H + between the active site and the reaction medium as shown in Eqn (1) and (2). E-Zn2'-OH-+ C 0 2 E-Zn2+-HCO; n20(1 1 (2) J E-Zn2+-OH2 + HCO; E-Zn2+-OHz + E-Zn2+-OH-+ H + .In carbonic anhydrase 11, reaction 2 occurs in two steps. First, a rate-limiting intramolecular proton transfer between metalbound water and His-64 followed by proton transfer between His-64 and buffer as indicated in Eqn (3).His-E-Zn2+-OHz + B + +H-His-E-Zn2+-OH-+ B + His-E-Zn2+-OH-+ BH'. (3)At pH 8, the C 0 2 hydration activity (kCa,/Km) of the muscle-specific carbonic anhydrase 111 is only 0.3% of that of Correspondence to Sven Lindskog, Avdelningen for Biokemi, Enzyme. Carbonic anhydrase (EC 4.2.1.1).Umei Universitet, S-90187 Umeb, Sweden isoenzyme I1 [2]. Whi...

Inhibition of the Archaeal β-Class (Cab) and γ-Class (Cam) Carbonic Anhydrases

“…2-3) [16,21,22,32,33,35,39,[41][42][43]45,48,50]. In the wellcharacterized -class, hydrogen bonded residues Glu106 and Thr199 further hydrogen bonds with the metal-bound hydroxide via the Thr199 O atom (Fig.…”

“…Although the five classes of CAs evolved independent from one another, kinetic analyses have shown three CA classes ( -, -and -) share the same two-step ping pong mechanism for the reversible hydration of carbon dioxide to bicarbonate (eq.2-3) [6,7,12,18,21,31,[34][35][36][37][38][39][40][41][42][43][44][45].…”

Prokaryotic carbonic anhydrases

“…This result differs with those for the α class carbonic anhydrases. Co substitution of the active site Zn in human CA II resulted in an enzyme with approximately 50% of the CO 2 hydration activity [144,145] even though X‐ray crystallography showed that the metal ion in the Co‐substituted human CA II has a tetrahedral coordination similar to Zn [146]. The possibility that the active site metal of authentic Cam from M. thermophila is Co instead of Zn remains to be determined.…”

Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III

“…This is demonstrated for three mutants of HCA III in Figure 2. The values of wild-type HCA III and T199S HCA III could not be determined because of denaturation of the enzyme at low pH; this for CA III has been estimated at 5.0 on the basis of spectroscopic and inhibition data for cobalt(II)-substituted bovine CA III (Ren et al, 1988a). The replacement Thr-199 -* Ser in HCA III did not place the apparent into the pH range of these measurements.…”